Figure 5
Figure 5. Reduced KIT receptor internalization in p85β-deficient BMMCs compared with WT in response to SCF. (A) WT or P85β−/− BMMCs were starved overnight and incubated with cycloheximide (100 ng/mL) for 2 hours. After cycloheximide treatment, cells were stimulated with SCF (100 ng/mL) for indicated time points, and KIT receptor internalization was studied by staining the cells with PE-conjugated anti-KIT receptor antibody followed by flow cytometric analysis. Shown is one of 4 independent experiments performed in triplicate. *P < .01, WT versus P85β−/−. (B) KIT receptor internalization is enhanced in cells expressing p85β compared with p85α on SCF stimulation. Cells (32D) coinfected with KIT and p85α or p85β were starved for 8 hours and incubated with cycloheximide (100 ng/mL) for 2 hours. After cycloheximide treatment, cells were stimulated with SCF (100 ng/mL) for indicated time points, and KIT receptor internalization was studied by staining the cells with PE-conjugated anti-KIT receptor antibody followed by flow cytometric analysis. Shown is one of 6 independent experiments performed in triplicate. *P < .01, p85α versus p85β. (C) Enhanced KIT receptor degradation in cells expressing p85β compared with p85α on SCF stimulation. Cells (32D) expressing WT KIT and p85α or p85β subunits were starved for 8 hours and incubated with cycloheximide (100 ng/mL) for 2 hours. After cycloheximide treatment, cells were stimulated with SCF (100 ng/mL) for indicated time points, and equal amount of protein lysates were subjected to Western blot analysis using an anti-KIT receptor antibody. Similar results were observed in 4 independent experiments. (D) p85β preferentially binds to c-Cbl compared with p85α in response to SCF stimulation. 32D cells coinfected with KIT and p85α or p85β were starved for 8 hours and stimulated with SCF (100 ng/mL) for 5 minutes. Equal amount of cell lysates (500 μg) were immunoprecipitated with an anti-HA antibody followed by Western blotting with a phospho-c-Cbl antibody. Data are from one of 4 independent experiments.

Reduced KIT receptor internalization in p85β-deficient BMMCs compared with WT in response to SCF. (A) WT or P85β−/− BMMCs were starved overnight and incubated with cycloheximide (100 ng/mL) for 2 hours. After cycloheximide treatment, cells were stimulated with SCF (100 ng/mL) for indicated time points, and KIT receptor internalization was studied by staining the cells with PE-conjugated anti-KIT receptor antibody followed by flow cytometric analysis. Shown is one of 4 independent experiments performed in triplicate. *P < .01, WT versus P85β−/−. (B) KIT receptor internalization is enhanced in cells expressing p85β compared with p85α on SCF stimulation. Cells (32D) coinfected with KIT and p85α or p85β were starved for 8 hours and incubated with cycloheximide (100 ng/mL) for 2 hours. After cycloheximide treatment, cells were stimulated with SCF (100 ng/mL) for indicated time points, and KIT receptor internalization was studied by staining the cells with PE-conjugated anti-KIT receptor antibody followed by flow cytometric analysis. Shown is one of 6 independent experiments performed in triplicate. *P < .01, p85α versus p85β. (C) Enhanced KIT receptor degradation in cells expressing p85β compared with p85α on SCF stimulation. Cells (32D) expressing WT KIT and p85α or p85β subunits were starved for 8 hours and incubated with cycloheximide (100 ng/mL) for 2 hours. After cycloheximide treatment, cells were stimulated with SCF (100 ng/mL) for indicated time points, and equal amount of protein lysates were subjected to Western blot analysis using an anti-KIT receptor antibody. Similar results were observed in 4 independent experiments. (D) p85β preferentially binds to c-Cbl compared with p85α in response to SCF stimulation. 32D cells coinfected with KIT and p85α or p85β were starved for 8 hours and stimulated with SCF (100 ng/mL) for 5 minutes. Equal amount of cell lysates (500 μg) were immunoprecipitated with an anti-HA antibody followed by Western blotting with a phospho-c-Cbl antibody. Data are from one of 4 independent experiments.

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